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LncRNA研究案例
發布日期:2015-04-27 17:01:31 瀏覽次數 :
導讀
相對于蛋白編碼序列以及小分子RNA,lncRNA的研究還僅僅只是處于起步階段,其功能與調控機制仍有待進一步闡明。

案例一:lncRNA- PACER激活致癌基因COX-2表達

作用類型:轉錄調控/表觀遺傳調控

研究者懷疑致癌基因COX-2的轉錄控制與ncRNA有關,使用人乳腺表皮細胞(HMEC)進行ChIP實 驗,在COX-2轉錄起始位點(TSS)上游-0.3kb處,發現一個額外啟動子,且這個啟動子和COX-2啟動子內有兩個CTCF/cohesin二聚 體的結合位點。沉默CTCF/cohesin,ChIP分析COX-2啟動子及上游區域內染色質修飾水平,發現這兩個二聚體所隔離出的染色質區域轉錄產生 了一段lncRNA,研究者將此lncRNA命名為p50-Associated COX-2 Extragenic RNA,PACER)。對沉默PACER后的HMEC進行ChIP-qPCR實驗,證實PACER可富集組蛋白乙酰基轉移酶p300,促進染色質乙酰基 化,影響RNA pol II復合物形成(RNAP II)。不過,RIP-qPCR發現PACER并非直接與p300互作,RIP及RNA蛋白互作實驗證明NF-κB家族蛋白p50會與PACER結 合,PACER則會促進結合在基因啟動子上的基因抑制復合物p50/p50二聚體轉化成p50/p65二聚體,實現富集p300的作用。

 
ChIP-Seq發現Cox-2 TSS上游有額外啟動子


ChIP確認兩個啟動子區域內有CTCF/cohesin結合位點


ChIP驗證CTCF沉默對不同組蛋白修飾的影響


RIP驗證p50同PACER互作


PACER作用通路示意圖

相關文獻:Krawczyk M, Emerson B M. p50-associated COX-2 extragenic RNA (PACER) activates COX-2 gene expression by occluding repressive NF-κB complexes[J]. eLife, 2014, 3.
 
 
案例二:lncRNA拷貝數變異引發癌癥

作用類型:轉錄調控/突變

本項研究中,研究者將目光集中于lncRNA拷貝數變異同癌癥發生的關系上,他們分析了不同數據庫中癌癥樣 本的SNP檢測結果,,篩選出188個出現SCNA的lncRNA,使用shRNA在數個癌細胞系中沉默這些lncRNA,發現在沉默FAL1基因 (focally amplified lncRNA on chromosome 1)時在體內和體外都會顯著減少癌細胞數目,將此lncRNA的cDNA導入卵巢表皮細胞,也會誘導細胞增殖,且在對128個卵巢癌晚期病人癌細胞進行檢 測后,發現FAL1的獲得性SCNA與病人存活率降低有關。
為了探明FAL1獲得性SCNA的致癌機制,研究者首先檢測了更多癌細胞中FAL1 RNA轉錄情況,結果發現,FAL1 RNA轉錄同獲得性SCNA為正向相關。使用RNA pull down、Western Bloting及RIP qPCR確認FAL1 RNA的結合蛋白為BMI1。由于BMI1又是PRC1(polycomb repressive complex 1)復合體的一個亞基,FAL1對BMI1的穩定又會進一步提高PRC1的穩定性。PRC1是一個染色體修飾復合物,會抑制很多基因表達。在癌細胞中用 shRNA沉默FAL1或BMI1,發現表達量上升最大的是CDKN1A基因,該基因與癌癥發生有關,而FAL1可能是通過調控BMI1在CDKN1A啟 動子的結合效率來控制CDKN1A的轉錄,提高CDKN1A表達量。另外,沉默FAL1會明顯誘導癌細胞進入G0/G1期,增加細胞衰老速度,也進一步顯 示了FAL1的致癌作用。



 
RNA pull down流程及結果


 
 
RIP檢測過程與結果


相關文獻:Hu X, Feng Y, Zhang D, et al. A Functional Genomic Approach Identifies FAL1 as an Oncogenic Long Noncoding RNA that Associates with BMI1 and Represses p21 Expression in Cancer[J]. Cancer cell, 2014, 26(3): 344-357.
 
案例三:lncRNA lnc-DC控制人類樹突狀細胞分化

lncRNA作用類型:作為配體保護轉錄因子STAT3磷酸化位點

研究者利用外周血單核細胞誘導分化為樹突狀細胞這一模型,通過轉錄組芯片及RNA-Seq,首先鑒別出一個 在人DC中特異表達的lncRNA,并將其命名為lnc-DC。后利用ChIP-Seq確認lnc-DC基因轉錄起始位點處H3K4me3組蛋白修飾水平 上升,轉錄調控因子PU.1親和力增加,確定DC分化過程中lnc-DC上調原因。

其后,研究者使用慢病毒載體沉默DC中的lnc-DC,使用轉錄組芯片及流式細胞術分析,發現DC功能基因下調,多種T細胞激活關鍵蛋白下調,單核細胞標記上調。說明lnc-DC的缺失會改變DC分化趨勢,影響DC功能。

通過RNA FISH確認lnc-DC定位于核內,RIP-Seq發現lnc-DC不會與AGO2和ALU元件結合。研究者使用生物素化lnc-DC,通過RNA pull down及MS分析,確認lnc-DC與轉錄因子STAT3互作。使用lnc-DC突變體及攜帶不同標記的STAT3分別進行RIP實驗,確認lnc- DC與STAT3結合模式。最后通過免疫印跡、核內外免疫熒光檢測及磷酸化分析,發現lnc-DC會阻止磷酸酶SHP1與STAT3的結合,保護Y705 磷酸化狀態,增強DC中的STAT3信號通路,以此實現其維持與促進人DC發育成熟和激活T細胞免疫應答的能力,最終對DC分化發育、抗原遞呈與免疫激活 功能起到至關重要的作用。


lnc-DC基因ChIP-Seq (上面三行)多位點ChIP-qPCR(中間兩行)結果


lnc-DC/STAT3免疫印跡和RIP結果


 
lncRNA芯片數據



 
lnc-DC在人DC分化過程中作用模式圖

相關文獻:Wang P, Xue Y, Han Y, et al. The STAT3-binding long noncoding RNA lnc-DC controls human dendritic cell differentiation[J]. Science, 2014, 344(6181): 310-313.
 
案例四:lncRNA通過超保守區域影響Drosha剪切產生成熟miR-195

作用類型:轉錄后調控/miRNA調控

miR-195已經證明與癌癥細胞增殖與耐藥性有關,在本項研究中,研究者希望進一步了解miR-195的 調控機制,他們利用miRNA芯片檢測HCT116細胞,通過分析差異miRNA種子序列,發現Uc.283+A這個lncRNA中的超保守區域(T- UCR)與pri-miR-195上Drosha剪切位點上游存在一段互補序列。EMSA實驗證明pri-miR-195確實會同Uc.283+A互作。 使用qPCR檢測HCT116細胞和SK-N-BE(2)細胞中Uc.283+A與成熟miR-195表達狀態,發現二者負相關;對其他數個類型癌細胞進 行脫甲基化處理后發現,Uc.283+A、pri-miR-195和成熟miR-195中CpG島被脫甲基化后,Uc.283+A、pri-miR- 195會上調,但miR-195卻會下調。使用野生型Uc.283+A與pri-miR-195進行體外孵育實驗,Uc.283+A與成熟miRNA依然 呈負相關,但如果使用pri-miR-195突變體或兩個RNA互補區結合阻礙物進行孵育,成熟miR-195則不再受影響。

以上實驗充分證明,Uc.283+A是利用T-UCR中與pri-miR-195的互補序列,下調成熟 miR-195。因為兩ncRNA互補區位于Drosha剪切位點上游,研究者假設這個過程是通過兩RNA互補區結合影響Drosha對pri-miR- 195的剪切。為此,研究者使用RNA antisense purification(RAP)法,發現兩RNA之間的互補配對,會影響一個名為DGCR8的蛋白同pri-miR-195的結合,由于Drosha 需要同DGCR8蛋白形成復合體才能對pri-miR-195進行剪切,Uc.283+A對DGCR8的影響會直接阻礙Drosha-DGCR8復合體形 成,最終影響成熟miR-195的豐度。


Uc.283+A控制Drosha對pri-miR-195剪切機制示意圖


 
RNA antisense purification(RAP)示意圖


相關文獻:Liz J, Portela A, Soler M, et al. Regulation of pri-miRNA Processing by a Long Noncoding RNA Transcribed from an Ultraconserved Region[J]. Molecular Cell, 2014.

案例五:HOTAIR改變染色質狀態促進癌癥轉移

作用類型:表觀遺傳調控

通過超高密度tiling array及qPCR分析不同乳腺癌細胞(原發性乳腺癌細胞和轉移型乳腺癌細胞),發現HOTAIR顯著上調。沉默HOTAIR,通過活體成像可以明顯發 現癌細胞轉移減弱。HOTAIR通常會與組蛋白H3K27甲基化酶復合體PRC2結合,發揮調控作用,通過ChIP-on-chip,發現在HOTAIR 上調時,有854個基因被PRC2所結合,出現H3K27me3組蛋白修飾,許多基因都是癌癥轉移的正向調節因子,也說明HOTAIR會通過與PRC2互 作,改變染色質狀態促進癌癥轉移。


 
ChIP-on-chip結果



 
HOTAIR-PRC2互作,改變組蛋白修飾狀態,影響癌癥基因表達

相關文獻:Gupta R A, Shah N, Wang K C, et al. Long non-coding RNA HOTAIR reprograms chromatin state to promote cancer metastasis[J]. Nature, 2010, 464(7291): 1071-1076.

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